Abstract
Low transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037). Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22 °C and 25 °C, and a 400 μM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciens overgrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future.
Highlights
Wheat (Triticum aestivum) is one of the most cultivated cereal crops worldwide and is a major source of protein and carbohydrates to humans
The only known protocol giving high stable transformation efficiency is from Japan Tobacco (Ishida et al, 2015), for which use is permitted under license payment
Nothing is known about the liability of Brazilian genotypes for genetic transformation mediated by A. tumefaciens, but there are great interests in using genetic material presenting good agronomic traits
Summary
Wheat (Triticum aestivum) is one of the most cultivated cereal crops worldwide and is a major source of protein and carbohydrates to humans. We attempted to adjust the factors responsible for the induction of A. tumefaciens virulence by determining the best combinations of pH, temperature and phenolic compound (AS) for T-DNA delivery into wheat genomes, as well as its effect on the regeneration of immature embryos subjected to the transformation processes.
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